Copolymer 1 related polypeptides for use as molecular weight markers and for therapeutic use

ABSTRACT

The present invention provides processes for determining the molecular weight of glatiramer acetate and other copolymers using molecular weight markers. The present invention further provides a plurality of molecular weight markers for determining the molecular weight of glatiramer acetate and other copolymers which display linear relationships between molar ellipticity and molecular weight, and between retention time and the log of the molecular weight. The molecular weight markers also optimally demonstrate biological activity similar to glatiramer acetate or corresponding copolymers and can be used for treating or preventing various immune diseases.

RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 09/816,989,filed Mar. 23, 2001, now U.S. Pat. No. 6,800,287, issued Oct. 5, 2004,which is a continuation of PCT International Application No.PCT/US99/22402, filed Sep. 24, 1999, which claims the benefit of U.S.Provisional Application Nos. 60/101,825 and 60/101,693, both filed Sep.25, 1998, which are incorporated by reference herein.

INTRODUCTION

The present invention provides molecular weight markers for accuratedetermination of the molecular weight of glatiramer acetate, terpolymersand other copolymers. The molecular weight markers are polypeptideshaving identified molecular weights between about 2,000 daltons andabout 40,000 daltons, and an amino acid composition corresponding toglatiramer acetate or a related copolymer. Identified molecular weightsare provided by polypeptides having defined sequences. Molecular weightmarkers corresponding to glatiramer acetate comprise the amino acidsalanine, glutamic acid, tyrosine and lysine in specific molar ratios.Molecular weight markers corresponding to related terpolymers comprisethree of the four amino acids. In a preferred embodiment, thepolypeptide has alanine at the N-terminus and tyrosine at the fourthposition from the N-terminus. The present invention further provides aplurality of molecular weight markers for determining the molecularweight range of a copolymer composition. The plurality of molecularweight markers ideally displays linear relationships between molarellipticity and molecular weight, or between retention time and the logof molecular weight.

Optimally, the polypeptides demonstrate biological activity similar tothe copolymer from which they are derived. Polypeptides having definedmolecular weights and amino acid compositions similar to glatirameracetate optimally have therapeutic utility for the treatment of immunediseases and conditions.

BACKGROUND OF THE INVENTION

Autoimmune diseases occur when an organism's immune system fails torecognize some of the organism's own tissues as “self” and attacks themas “foreign.” Normally, self-tolerance is developed early bydevelopmental events within the immune system that prevent theorganism's own T cells and B cells from reacting with the organism's owntissues. These early immune responses are mediated by the binding ofantigens to MHC molecules and presentation to T cell receptors.

This self-tolerance process breaks down when autoimmune diseases developand the organism's own tissues and proteins are recognized as“autoantigens” and attacked by the organism's immune system. Forexample, multiple sclerosis is believed to be an autoimmune diseaseoccurring when the immune system attacks the myelin sheath, whosefunction is to insulate and protect nerves. It is a progressive diseasecharacterized by demyelination, followed by neuronal and motor functionloss. Rheumatoid arthritis (“RA”) is also believed to be an autoimmunedisease which involves chronic inflammation of the synovial joints andinfiltration by activated T cells, macrophages and plasma cells, leadingto a progressive destruction of the articular cartilage. It is the mostsevere form of joint disease. The nature of the autoantigen(s) attackedin rheumatoid arthritis is poorly understood, although collagen type IIis a candidate.

A tendency to develop multiple sclerosis and rheumatoid arthritis isinherited. These diseases occur more frequently in individuals carryingone or more characteristic MHC class II alleles. For example, inheritedsusceptibility for rheumatoid arthritis is strongly associated with theMHC class II DRB1 *0401, DRB 1 *0404, or DRB 1*0405 or the DRB1*0101alleles. The histocompatibility locus antigens (HLA) are found on thesurface of cells and help determine the individuality of tissues fromdifferent persons. Genes for histocompatibility locus antigens arelocated in the same region of chromosome 6 as the majorhistocompatibility complex (MHC). The MHC region expresses a number ofdistinctive classes of molecules in various cells of the body, the genesbeing, in order of sequence along the chromosome, the Class I, II andIII MHC genes. The Class I genes consist of HLA genes, which are furthersubdivided into A, B and C subregions. The Class II genes are subdividedinto the DR, DQ and DP subregions. The MHC-DR molecules are the bestknown; these occur on the surfaces of antigen presenting cells such asmacrophages, dendritic cells of lymphoid tissue and epidermal cells. TheClass III MHC products are expressed in various components of thecomplement system, as well as in some non-immune related cells.

A number of therapeutic agents have been developed to treat autoimmunediseases, including steroidal and non-steroidal anti-inflammatory drugs,for example, methotrexate; various interferons; and certain inhibitorsof prostaglandin synthesis. However, these agents can be toxic when usedfor more than short periods of time or cause undesirable side effects.Other therapeutic agents bind to and/or inhibit the inflammatoryactivity of tumor necrosis factor (TNF), for example, anti-TNF specificantibodies or antibody fragments, or a soluble form of the TNF receptor.These agents target a protein on the surface of a T cell and generallyprevent interaction with an antigen presenting cell (APC). However,therapeutic compositions containing natural folded proteins are oftendifficult to produce, formulate, store, and deliver. Moreover, theinnate heterogeneity of the immune system can limit the effectiveness ofdrugs and complicate long-term treatment of autoimmune diseases.

Glatiramer acetate (Copolymer 1; Cop 1; hereinafter GLAT copolymer) is amixture of polypeptides composed of alanine, glutamic acid, lysine, andtyrosine in a molar ratio of approximately 4.6:1.5:3.6:1.0,respectively, which is synthesized by chemically polymerizing the fouramino acids, forming products with average molecular weights rangingfrom about 4000 to about 13,000 daltons. The corresponding molarfractions are approximately 0.427 for alanine, 0.141 for glutamic acid,0.337 for lysine and 0.093 for tyrosine, and may vary by about +/−10%.Related copolymers are mixtures of polypeptides composed of three (thus,“terpolymers”) of the four aforementioned amino acids. Copolymer 1 andthe terpolymers address the innate heterogeneity of the mammalian immunesystem and human population and are effective for treatment ofautoimmune diseases and other immune conditions. Preferred averagemolecular weight ranges and processes of making terpolymers aredescribed in U.S. Pat. No. 5,800,808, which is hereby incorporated byreference in its entirety. Copolymer-1, according to the presentinvention, may be prepared by methods known in the art, for example, theprocess disclosed in U.S. Pat. No. 3,849,550, wherein theN-carboxyanhydrides of tyrosine, alanine, γ-benzyl glutamate andE-N-trifluoro-acetyllysine are polymerised at ambient temperature inanhydrous dioxane with diethylamine as initiator. The deblocking of theγ-carboxyl group of the glutamic acid is effected by hydrogen bromide inglacial acetic acid and is followed by the removal of thetrifluoroacetyl groups from the lysine residues by 1M piperidine. Forthe purposes of the application, the terms “ambient temperature” and“room temperature” should be understood to mean a temperature rangingfrom about 20° to about 26° C. The copolymer-1 with the requiredmolecular weight profile can be obtained either by methods known per se.Such methods include chromatography of copolymer-1 containing highmolecular weight species and collecting the fractions without theundesired species or by partial acid or enzymatic hydrolysis to removethe high molecular weight species with subsequent purification bydialysis or ultrafiltration. A further method to obtain copolymer-1 withthe desired molecular weight profile is by preparing the desired specieswhile the amino acids are still protected and then obtain the correctspecies directly upon removing the protection. The compositions of thepresent invention may be formulated by conventional methods known in theart. Preferably, the composition is lyophilized and formed into anaqueous solution suitable for sub-cutaneous injection. Alternatively,copolymer-1 may be formulated in any of the forms known in the art forpreparing oral, nasal, buccal, or rectal formulations of peptide drugs.Also contemplated by the invention are other copolymers comprised ofother combinations of three, four, or five or more amino acids.

To certify a Copolymer 1 or terpolymer preparation for use in apharmaceutical products, it is necessary to accurately determine themolecular weight distribution of the polypeptides in the preparation.One method for determining the molecular weight is chromatography on aSuperose 12 column (a cross-linked, agarose-based medium with anexclusion limit of 2×10⁶ Daltons, an optimal separation range of 1000 to3×10⁵ Daltons, and a bead diameter of 20–40 μm) . Calibrationcoefficients of columns for determination of glatiramer acetatemolecular weight have been determined using glatiramer acetate batcheswith indirectly measured molecular weights. Indirect measures haveincluded viscosimetry and velocity-sedimentation ultracentrifugation.More recently, batches of glatiramer acetate markers have been preparedwhose molecular weights were determined by multiple angle laser lightscattering (MALLS).

Thus, a need exists for molecular weight markers useful as standards fordetermining the molecular weight distribution of copolymer compositionscontemplated by the invention. Desirable molecular weight markers havedefined molecular weights and physical properties which are analogous tothe molecules for which molecular weight is to be determined. Ideally,there is a linear relationship between the defined molecular weights (orthe log of the defined molecular weights) of the markers and ameasurable physical property such as, for example, the molar ellipticityof the markers, or the retention time of the markers on a molecularsizing column.

SUMMARY OF THE INVENTION

Sequence-defined molecular weight markers that have chemical andphysical characteristics similar to GLAT copolymer provide an accurateand robust calibration set for determinations of molecular weight ofproduction batches. The present invention provides derivatives of GLATcopolymer useful as molecular weight markers for determining themolecular weight ranges of GLAT copolymer preparations and optimallyhaving therapeutic utility for treatment of immune conditions. Theinvention further provides polypeptides having defined molecular weightswhich are derivatives of other copolymers and which are useful fordetermining molecular weight ranges of preparations of those copolymers.When those copolymers are therapeutically useful, the derivativepolypeptides optimally have therapeutic utility. For determination ofthe molecular weight range of a GLAT copolymer preparation, thepreferred derivative is a polypeptide having an amino acid compositoncorresponding approximately to GLAT copolymer and an identifiedmolecular weight which is between about 2,000 daltons and about 40,000daltons. The polypeptide preferably has specific molar ratios of aminoacids alanine, glutamic acid tyrosine and lysine. Moreover, in apreferred embodiment the polypeptide has alanine at the N-terminus andtyrosine at the fourth position from the N-terminus. For determinationof the molecular weight of a terpolymer, the preferred derivative willhave a defined molecular weight and an amino acid compositioncorresponding approximately to that of the terpolymer. Other copolymersare also contemplated by the invention. When determining of themolecular weight of a copolymer contemplated by the invention, thepolypeptide derivative will have a defined molecular weight and an aminoacid composition corresponding approximately to that of the copolymer.

The present invention further provides a plurality of molecular weightmarkers for determining the molecular weight of glatiramer acetate or aterpolymer or other copolymer on a molecular weight sizing column. Themarkers comprise two to ten or more polypeptides, each polypeptidehaving an identified molecular weight. When determining the molecularweight range of glatiramer acetate, a preferred plurality of molecularweight markers will have defined molecular weights from about 2,000daltons to about 40,000 daltons, and amino acid compositionscorresponding to glatiramer acetate or a selected terpolymer. Inpreferred embodiments, there is a linear relationship between the logmolecular weight of the polypeptide molecular weight markers and eitherthe retention time of the molecular weight markers on a sizing column orbetween the molecular weight of the molecular weight markers and themolar ellipticity of the molecular weight markers.

The present invention further provides pharmaceutical compositions whichinclude a therapeutically effective amount of a polypeptide useful as amolecular weight marker for determining the molecular weight range ofGLAT copolymer and consisting essentially of amino acids alanine,glutamic acid, tyrosine and lysine in molar fractions of from about 0.38to about 0.50 alanine, from about 0.13 to about 0.15 glutamic acid, fromabout 0.08 to about 0.10 tyrosine, and from about 0.3 to about 0.4lysine, and a pharmaceutically acceptable carrier.

The present invention further provides pharmaceutical compositions whichinclude a therapeutically effective amount of a polypeptide useful as amolecular weight marker for determining the molecular weight range of aterpolymer and consisting essentially of amino acids alanine, tyrosine,and lysine in the molar fractions of from about 0.3 to about 0.6alanine, from about 0.005 to about 0.25 tyrosine, and from about 0.1 toabout 0.5 lysine, and a pharmaceutically acceptable carrier. Thepolypeptide is preferably substantially free of glutamic acid.

The present invention further provides pharmaceutical compositions whichinclude a therapeutically effective amount of a polypeptide useful as amolecular weight marker for determining the molecular weight range of aterpolymer and consisting essentially of glutamic acid, tyrosine andlysine in molar fractions of from about 0.005 to about 0.300 glutamicacid, from about 0.005 to about 0.250 tyrosine, and from about 0.3 toabout 0.7 lysine, and a pharmaceutically acceptable carrier. Thepolypeptide is preferably substantially free of alanine.

The present invention further provides pharmaceutical compositions whichinclude a therapeutically effective amount of a polypeptide useful as amolecular weight marker for determining the molecular weight range of aterpolymer and consisting essentially of amino acids alanine, glutamicacid and tyrosine in molar fractions of from about 0.005 to about 0.8alanine, from about 0.005 to about 0.3 glutamic acid, and from about0.005 to about 0.25 tyrosine, and a pharmaceutically acceptable carrier.The polypeptide is preferably substantially free of lysine.

The present invention also provides pharmaceutical compositions whichincludes a therapeutically effective amount of a polypeptide useful as amolecular weight marker for determining the molecular weight range of aterpolymer and consisting essentially of alanine, glutamic acid andlysine, in molar fractions of from about 0.005 to about 0.6 alanine,from about 0.005 to about 0.3 glutamic acid, and from about 0.2 to about0.7 lysine, and a pharmaceutically acceptable carrier. The polypeptideis preferably substantially free of tyrosine.

In general, pharmaceutical compositions of the invention includetherapeutically effective amounts of a polypeptide which is useful as amolecular weight marker for determining the molecular weight range of acopolymer of any number (e.g., three to five or more) of amino acids. Inthe manner of glatiramer acetate, such a copolymer is a diversepopulation of sequences of the amino acids. The polypeptide useful as amolecular weight marker consists of those amino acids in molar fractionscorresponding approximately to the copolymer.

The present invention further provides methods for treating andpreventing immune-mediated and autoimmune diseases in a mammal whichinclude administering a therapeutically effective amount of a molecularweight marker of the invention. In another embodiment, the method fortreating immune-mediated and autoimmune diseases in a mammal furtherinvolves inhibiting proliferation of T cells involved in the immuneattack. In another embodiment, the method for treating immune-mediatedand autoimmune diseases in a mammal involves binding a molecular weightmarker of the invention to an antigen presenting cell. In yet anotherembodiment, the method for treating immune-mediated and autoimmunedisease in a mammal involves binding a molecular weight marker of theinvention to a major histocompatibility complex class II protein whichis associated with autoimmune diseases.

Autoimmune diseases contemplated by the present invention includearthritic conditions, demyelinating diseases and inflammatory diseases.For example, autoimmune diseases which can be treated by the presentcompositions include multiple sclerosis, rheumatoid arthritis,osteoarthritis, autoimmune hemolytic anemia, autoimmune oophoritis,autoimmune thyroiditis, autoimmune uveoretinitis, Crohn's disease,chronic immune thrombocytopenic purpura, colitis, contact sensitivitydisease, diabetes mellitus, Graves disease, Guillain-Barre's syndrome,Hashimoto's disease, idiopathic myxedema, myasthenia gravis, psoriasis,pemphigus vulgaris, or systemic lupus erythematosus.

Immune-mediated diseases result from undesired sensitivity of the immunesystem to particular foreign antigens. Examples are host-versus-graftdisease (HVGD) and graft-versus-host disease (GVHD) and numerous typesof delayed-type hypersensitivity (DTH).

The present compositions can be used to treat one or more of thesediseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a-1, 1 a-2 and 1 a-3 provide[s] the distribution of alanine inthe molecular markers (TV-markers) described in Table 1. The amino acidposition is defined by the X-axis. The presence of an alanine isindicated by a vertical bar at the indicated amino acid position.

FIGS. 1 b-1, 1 b-2 and 1 b-3 provide[s] the distribution of lysine inthe TV-markers described in Table 1. The amino acid position is definedby the X-axis. The presence of a lysine residue is indicated by avertical bar at the indicated amino acid position.

FIGS. 1 c-1, 1 c-2 and 1 c-3 provide[s] the distribution of glutamicacid in the TV-markers described in Table 1. The amino acid position isdefined by the X-axis. The presence of a glutamic acid residue isindicated by a vertical bar at the indicated amino acid position.

FIGS. 1 d-1, 1 d-2 and 1 d-3 provide[s] the distribution of tyrosine inthe TV-markers described in Table 1. The amino acid position is definedby the X-axis. The presence of a tyrosine residue is indicated by avertical bar at the indicated amino acid position.

FIG. 2 provides a plot of the molar ellipticity versus molecular weightof the present TV-markers compared to known glatiramer acetate markers.The molar ellipticity is provided in 10⁻⁵ deg cm⁻² dmole⁻¹ and themolecular weight is in daltons. Circles indicate TV-markers and squaresdepict glatiramer acetate markers. As shown, the TV-markers provide alinear relationship between molar ellipticity and molecular weight.

Ellipticity of TV-markers and the currently use glatiramer acetatemolecular weight markers as a function of their molecular weight. Theexperimental CD values are presented below.

FIG. 3 a provides a plot of the relative retention time (RRT) of thepresent TV-markers versus the log molecular weight of those markers,using the RRT-based algorithm.

FIG. 3 b provides a plot of the log molecular weight of the TV-markersversus the retention time (RT) of those markers, using theMillennium-based algorithm.

FIG. 4 a provides a plot summarizing several calibrations of therelative retention time (RRT) of the present TV-markers versus themolecular weight of those markers, using the RRT-based algorithm. Datawere obtained from sixteen columns. Average values for each of thesixteen calibrations are depicted.

FIG. 4 b provides a plot summarizing several calibrations of themolecular weight of the TV-markers versus the relative retention time(RRT) of those markers, using the Millennium-based algorithm. Data wereobtained from sixteen columns. Average values for each of the sixteencalibrations are depicted.

Summary calibrations of TV-markers in two TEVA laboratories. Data wasobtained from 16 columns tested from Apr. 1997 to Feb. 1998. For each ofthe 16 columns tested the average values are used in the display. Thecalibrations are presented in the currently used RRT-algorithm (a) andthe Millennium-based algorithm (b)

FIG. 5 depicts inhibition of Cop 1 binding to anti-Cop 1 polyclonalantibodies by four TV-markers and Cop 1 (03494). Absorbance ratioindicates absorbance measured with increasing inhibitor concentrationrelative to absorbance in the absence of binding inhibition.

DETAILED DESCRIPTION OF THE INVENTION

Molecular weight markers of the invention (e.g., TV-markers), includepolypeptides having an amino acid composition approximatelycorresponding to glatiramer acetate or related terpolymers, and anidentified molecular weight which is between about 2,000 daltons andabout 40,000 daltons and are useful for accurately determining themolecular weight of GLAT copolymer and related terpolymers. It followsfrom the requirement for an identified molecular weight that a TV-markershould have a discrete molecular weight and not a range of molecularweights. Accordingly, TV-markers are synthesized according to apredetermined amino acid sequence which corresponds in composition tothe copolymer for which molecular weight range is to be determined.Optimally, TV-markers have therapeutic activity which is similar tocorresponding copolymer. These markers can be used in any molecular sizediscrimination system using any available molecular weight determinationprocedure or apparatus. For example, the present markers can be used forcalibration of any chromatographic procedure or apparatus which is usedfor molecular weight determinations of polypeptides or proteins. Such achromatographic apparatus can be a molecular weight sizing column whichseparates polypeptides on the basis of their molecular size. Examples ofmolecular weight sizing columns include TSK columns, Sephadex columns,Sepharose columns, and Superose columns. In order to provide molecularweight markers of discrete size and composition, molecular weightmarkers of the invention can be synthesized according to predeterminedsequences by methods which are well known to those of skill in the art.

Amino acids of the present invention include, but are not limited to the20 commonly occurring amino acids. Also included are naturally occurringand synthetic derivatives, for example, selenocysteine. Amino acidsfurther include amino acid analogues. An amino acid “analogue” is achemically related form of the amino acid having a differentconfiguration, for example, an isomer, or a D-configuration rather thanan L-configuration, or an organic molecule with the approximate size andshape of the amino acid, or an amino acid with modification to the atomsthat are involved in the peptide bond, so as to be protease resistantwhen polymerized in a peptide or polypepide.

The phrases “amino acid” and “amino acid sequence” as defined here andin the claims can include one or more components which are amino acidderivatives and/or amino acid analogs comprising part or the entirety ofthe residues for any one or more of the 20 naturally occurring aminoacids indicated by that sequence. For example, in an amino acid sequencehaving one or more tyrosine residues, a portion of one or more of thoseresidues can be substituted with homotyrosine. Further, an amino acidsequence having one or more non-peptide or peptidomimetic bonds betweentwo adjacent residues, is included within this definition.

The one letter and three letter amino acid codes (and the amino acidthat each represents) are as follows: A means ala (alanine); C means cys(cysteine); D means asp (aspartic acid); E means glu (glutamic acid); Fmeans phe (phenylalanine); G means gly (glycine); H means his(histidine); l means ile (isoleucine); K means lys (lysine); L means leu(leucine); M means met (methionine); N means asn (asparagine); P meanspro (proline); Q means gin (glutamine); R means arg (arginine); S meansser (serine); T means thr (threonine); V means val (valine); W means trp(tryptophan); and Y means tyr (tyrosine).

The term “hydrophobic” amino acid is defined here and in the claims asincluding aliphatic amino acids alanine (A, or ala), glycine (G, orgly), isoleucine (I, or ile), leucine (L, or leu), proline (P, or pro),and valine (V, or val), the terms in parentheses being the one letterand three letter standard code abbreviation s for each amino acid, andaromatic amino acids tryptophan (W, or trp), phenylalanine (F or phe),and tyrosine (Y, or tyr). The amino acids confer hydrophobicity as afunction of the length of aliphatic and size of aromatic side chains,when found as residues within a protein.

The term “charged” amino acid is defined here and in the claims asincluding an amino acids aspartic acid (D, or asp), glutamic acid (E, orglu), histidine (H, or his), arginine (R, or arg) and lysine (K, orlys), which confer a positive (his, lys and arg) or negative (asp andgly) charge at physiological values of pH in aqueous solutions onproteins containing these residues.

Polypeptide Compositions Contemplated by the Invention—According to thepresent invention, polypeptides having defined molecular weights andcomprising three or all four of the amino acids tyrosine, glutamic acid,alanine and lysine are preferred for the present markers. However, oneof skill in the art can readily substitute structurally-related aminoacids without deviating from the spirit of the invention. Thus, thepresent invention further contemplates conservative amino acidsubstitutions for tyrosine, glutamic acid, alanine and lysine in thepresent polypeptides. Such structurally-related amino acids includethose amino acids which have about the same charge, hydrophobicity andsize as tyrosine, glutamic acid, alanine or lysine. For example, lysineis structurally-related to arginine and histidine; glutamic acid isstructurally-related to aspartic acid; tyrosine is structurally-relatedto serine, threonine, tryptophan and phenylalanine; and alanine isstructurally-related to valine, leucine and isoleucine.

Moreover, molecular weight markers of the invention can be composed ofL- or D-amino acids. As is known by one of skill in the art, L-aminoacids occur in most natural proteins. However, D-amino acids arecommercially available and can be substituted for some or all of theamino acids used to make molecular weight markers of the invention. Thepresent invention contemplates molecular weight markers formed frommixtures of D- and L-amino acids, as well as molecular weight markersconsisting essentially of either L- or D-amino acids.

The average molecular weight and the average molar fraction of the aminoacids in the present polypeptides can vary. However, a molecular weightrange of about 2,000 to about 40,000 is contemplated, and basicpolypeptides, rather than acidic polypeptides, are preferred.

In one embodiment, the present invention provides polypeptide markerscontaining tyrosine, alanine, glutamic acid and lysine in defined molarratios. In a more preferred embodiment, the molar ratio of amino acidsof the present polypeptides is that found in GLAT copolymer. Such acorrespondence in molar ratios provides the best molecular weightmarkers because those markers will have a charge and a molecular shapewhich is similar to that of GLAT copolymer. When structurally dissimilarmarkers are used, the markers may migrate or elute somewhat differentlyfrom GLAT copolymer preparations, even though those preparations havethe same molecular weight as the markers.

Moreover, in a preferred embodiment, alanine is at the N-terminus andtyrosine is at position four from the N-terminus. Edman degradationanalyses performed on various glatiramer acetate batches revealed agreater abundance of alanine at the N-terminus and tyrosine at positionfour from the N-terminus. Therefore, in certain preferred embodiments,GLAT copolymer molecular weight markers have alanine at the N-terminusand tyrosine at position four from the N-terminus. Studies of thepolymerization reaction used to synthesize GLAT copolymer have indicatedthat alanine and glutamic acid polymerize faster than lysine. As aresult, the C-terminal portion of GLAT copolymer tends to be richer inalanine and glutamic acid, whereas the N-terminal portion tends to bericher in lysine. In preferred embodiments, the distribution of aminoacid residues in GLAT copolymer molecular weight markers reflects thisbias.

When determining the molecular weight range of GLAT copolymer, apreferred molecular weight marker consists essentially of amino acidsalanine, glutamic acid, tyrosine and lysine in molar fractions of fromabout 0.38 to about 0.50 alanine, from about 0.13 to about 0.15 glutamicacid, from about 0.08 to about 0.10 tyrosine, and from about 0.3 toabout 0.4 lysine.

In other embodiments, the present invention provides molecular weightmarkers containing three of the four amino acids alanine, glutamic acid,tyrosine, and lysine in defined ratios. In preferred embodiments, themolar fractions of amino acids present the molecular weight markerscorrespond to that found in a corresponding terpolymer.

When the molecular weight marker contains alanine, glutamic acid andtyrosine, alanine can be present in a mole fraction of about 0.005 toabout 0.800, glutamic acid can be present in a mole fraction of about0.005 to about 0.300, and tyrosine can be present in a mole fraction ofabout 0.005 to about 0.250. The molecular weight is from about 2,000 toabout 40,000 daltons, and preferably from about 3000 to about 12,000daltons.

When the molecular weight marker contains alanine, glutamic acid andlysine, alanine can be present in a mole fraction of about 0.005 toabout 0.600, glutamic acid can be present in a mole fraction of about0.005 to about 0.300, and lysine can be present in a mole fraction ofabout 0.2 to about 0.7. The molecular weight is between about 2,000 andabout 40,000 daltons, and preferably between about 3000 and about 12,000daltons.

When the molecular weight marker contains alanine, tyrosine and lysine,alanine can be present in a mole fraction of about 0.3 to about 0.6,tyrosine can be present in a mole fraction of about 0.005 to about0.250, and lysine can be present in a mole fraction of about 0.1 toabout 0.5. The molecular weight is between about 2,000 and about 40,000daltons, and preferably between about 3000 and about 12,000 daltons.

When the molecular weight marker contains glutamic acid, tyrosine andlysine, glutamic acid can be present in a mole fraction of about 0.005to about 0.300, tyrosine can be present in a mole fraction of about0.005 to about 0.250, and lysine can be present in a mole fraction ofabout 0.3 to about 0.7. The molecular weight is between about 2,000 andabout 40,000 daltons, and preferably between about 3000 and about 12,000daltons.

Polypeptides of the invention can be used for molecular weight rangedeterminations of other copolymers contemplated by the invention.Contemplated copolymers can consist of combinations of three, four, orfive or more amino acids. In general, in order to determine themolecular weight range of a copolymer contemplated by the invention, thepolypeptide molecular weight marker will have a defined molecular weightand an amino acid composition corresponding approximately to that of thecopolymer. It will be apparent to one of skill in the art that any biasin the distribution of amino acids in a copolymer can be determined asdescribed above for GLAT copolymer. For example, the relative amounts ofamino acids incorporated at each position of a terpolymer population canbe obtained by analyzing the products of each step of an Edmandegradation. Alternatively, the proportions of amino acids incorporatedinto a terpolymer population during synthesis can be monitored. Whereapplicable, molecular weight markers can then be synthesized whichreflect the bias. In addition, certain preferred terpolymer molecularweight markers will have alanine or tyrosine at position four.

Examples of preferred polypeptide molecular weight marker sequences aregiven in Table 1 (SEQ ID NOS: 1–7) using the conventional single letteramino acid code and reading from N-terminal to C-terminal. The sevenindicated sequences are individual preparations of polypeptides havingan amino acid composition corresponding to glatiramer acetate. Usually,amino acids comprising a molecular weight marker molecule arepredominantly of one configuration (D- or L-configuration). In preferredembodiments, a molecular weight marker molecule is composed entirely ofamino acids of the same configuration. However, molecular weight markermolecules comprising amino acids of mixed configuration may be preferredin certain embodiments where molecular weight is being determined for aglatiramer acetate preparation comprising amino acids of mixedconfiguration.

TABLE 1 Selected TV-markers amino acid sequences SEQ ID TV-## NOSequence TV-35 1 AKKYAKKEKAAKKAYKKEAKAKAAEAAAKEAAYEA TV-45 2AKKYAKKAKAEKAKKAYKAAEAKKAAKYEKAAAEKAAAKE- AAYEA TV-56 3AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAEAKY- KAEAAKAAAKEAAYEA TV-66 4AKKYAKKEKAYAKAKKAEAKAAKKAKAEAKKYAKAAKAEK- KEYAAAEAKYKAEAAKAAAKEAAYEATV-77 5 AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAKAEA-KKYAKAAKAEKKEYAAAEAKYKAEAAKAAAKEAAYEA TV-86 6AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAKAEA-KKYAKAAKAEKKEYAAAEAKYKAEAAKKAYKAEAAKAAAK- EAAYEA TV-109 7AKKYAKKAEKAYAKKAKAAKEKKAYAKKEAKAYKAAEAKK-KAKAEAKKYAKEAAKAKKEAYKAEAKKYAKAAKAEKKEYA- AAEAKKAEAAKAYKAEAAKAAAKEAAYEA

In another embodiment, the present invention provides a plurality ofmolecular weight markers for determining the molecular weight ofglatiramer acetate or a terpolymer on a molecular weight sizing column.The plurality of molecular weight markers are polypeptides. Theplurality of markers can be two to about ten or more. In a preferredembodiment, the plurality of markers is about seven. Each polypeptidehas an identified molecular weight which is between about 2,000 daltonsand about 40,000 daltons, and an amino acid composition whichcorresponds approximately to that of glatiramer acetate or a terpolymer.

When such a plurality of molecular weight markers are used as standardsfor determining the molecular weight of glatiramer acetate or aterpolymer, a relationship which is approximately linear exists betweenthe retention time of the molecular weight markers on thechromatographic column and the log of the molecular weight. A pluralityof markers is used which is sufficient to establish the approximatelylinear relationship, although more may be employed. FIG. 3 shows theapproximately linear relationship between relative retention time andlog molecular weight for TV-markers of the invention.

In another embodiment, an approximately linear relationship existsbetween the molar ellipticity of the molecular weight markers and themolecular weight of the markers. When determining the molecular weightof a glatiramer acetate preparation by molar ellipticity, a plurality ofmarkers is used which is sufficient to establish the approximatelylinear relationship, although more may be employed. A molecular weightfor the glatiramer acetate or terpolymer preparation is then obtainedbased on the linear relationship. FIG. 2 shows the approximately linearrelationship between molar ellipticity and molecular weight forTV-markers of the invention.

Pharmaceutical Compositions Contemplated by the Invention—Molecularweight markers of the invention which correspond in composition to GLATcopolymer optimally have biological activity, and can be used fortreatment of disease in the manner of GLAT copolymer. TV-markers havingbiological activity are alternately referred to as therapeutic markers.For example, GLAT copolymer is useful for the treatment of MS in humansas well as for blocking experimental allergic encephalomyelitis (EAE) inmice. Polypeptides of the invention having identified molecular weightsand amino acid compositions corresponding to GLAT copolymer are shownherein to be active in the mouse model as well and demonstrateimmunological characteristics which are similar to those of GLATcopolymer. Monoclonal antibodies which bind to GLAT copolymer also bindto TV-markers. Additionally, certain T cells which are stimulated byGLAT copolymer are also stimulated by molecular weight markers of theinvention.

Similarly, a polypeptide having a defined molecular weight andcorresponding in amino acid composition to a terpolymer havingtherapeutic utility will optimally have therapeutic utility. In general,polypeptide molecular weight markers corresponding in composition to abiologically active copolymer will optimally have similar biologicalactivity.

The present molecular weight markers can be formulated intopharmaceutical compositions containing a pharmaceutically acceptablecarrier. As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, sweetenersand the like. The pharmaceutically acceptable carriers may be preparedfrom a wide range of materials including, but not limited to, flavoringagents, sweetening agents and miscellaneous materials such as buffersand absorbents that may be needed in order to prepare a particulartherapeutic composition. The use of such media and agents withpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. The present compositions may be formulated as aninjectable solution or suspension, a spray solution or a suspension.

Pharmaceutical compositions comprise an amount of one or more molecularweight markers of the invention. Preferably, the molecular weightmarkers consist essentially of three or all four of the amino acidstyrosine, alanine, glutamic acid and lysine in defined molar fractions.The molar fractions of the amino acids will be as set forth above.

In one embodiment, the molecular weight markers of the pharmaceuticalcomposition are capable of binding to an MHC class II protein which,preferably, is associated with an autoimmune disease. The Class II MHCprotein consists of approximately equal-sized α and β subunits, both ofwhich are transmembrane proteins. A peptide-binding cleft is formed byparts of the amino termini of both α and β subunits. Thispeptide-binding cleft is the site of presentation of the antigen to Tcells. There are at least three types of Class II MHC molecules: HLA-DR,HLA-DQ, and HLA-DP molecules. There are also numerous alleles encodingeach type of these HLA molecules. The Class II MHC molecules areexpressed predominantly on the surfaces of B lymphocytes and antigenpresenting cells such as macrophages. Any available method can be usedto ascertain whether the molecular weight marker binds to one or moreMHC class II proteins. For example, the polypeptide can be radiolabeledor biotinylated, mixed with a crude or pure preparation of MHC class IIprotein and binding detected by adherence of the reporter molecule tothe MHC class II protein after removal of the unbound polypeptide.

In another embodiment, the molecular weight markers are capable ofbinding to an MHC class II protein associated with multiple sclerosis. Apolypeptide of this embodiment can have similar or greater affinity forthe antigen binding groove of an MHC class II protein associated withmultiple sclerosis than does Copolymer 1. Hence, the contemplatedpolypeptide can inhibit binding of or displace the binding of myelinautoantigens from the MHC class II protein. One MHC class II proteinassociated with multiple sclerosis is HLA-DR4 (DRB1*1501).

In another embodiment, molecular weight markers of the invention arecapable of binding to an MHC class II protein associated with anarthritic condition, for example, rheumatoid arthritis orosteoarthritis. Accordingly, a polypeptide of this embodiment can have agreater affinity for the antigen binding groove of an MHC class IIprotein associated with the autoimmune disease than does a type IIcollagen 261–273 peptide. Hence, the contemplated polypeptide caninhibit binding of, or displace the type II collagen 261–273 peptidefrom the antigen binding groove of an MHC class II protein.

Therapeutic Methods Contemplated by the Invention—The present inventionfurther provides methods for treating and preventing immune diseases ina mammal which include administering a therapeutically effective amountof a composition comprising a molecular weight marker of the invention.

Autoimmune diseases contemplated by the present invention include eithercell-mediated disease (e.g. T cell) or antibody-mediated (e.g. B cell)disorders. Such disorders can be, inter alia, arthritic conditions,demyelinating diseases and inflammatory diseases. For example,autoimmune diseases which can be treated by the present polypeptidesinclude multiple sclerosis (MS), rheumatoid arthritis (RA),osteoarthritis, autoimmune hemolytic anemia, autoimmune oophoritis,autoimmune thyroiditis, autoimmune uveoretinitis, Crohn's disease,chronic immune thrombocytopenic purpura, colitis, contact sensitivitydisease, diabetes mellitus, Graves disease, Guillain-Barre's syndrome,Hashimoto's disease, idiopathic myxedema, myasthenia gravis, psoriasis,pemphigus vulgaris, or systemic lupus erythematosus. The presentcompositions can be used to treat one or more of these diseases.

The term “arthritic condition” as used herein is a condition wherein atleast one symptom of rheumatoid arthritis is observed in at least onejoint of a mammal, for example in a shoulder, knee, hip, backbone or adigit of the mammal. Examples of arthritic conditions include“polyarthritis”, which is an arthritic condition that affects more thana single joint; “juvenile arthritis”, an arthritic condition of humansunder the age of 21; and Felty's syndrome, which can include thesymptoms of neutropenia, splenomegaly, weight loss, anemia,lymphadenopathy, and pigment spots on the skin.

Immune-mediated diseases contemplated by the present invention arecharacterized by undeisrable immune hypersensitivity to one or moreantigens and include host-versus-graft disease (HVGD) andgraft-versus-host disease (GVHD), which are exemplified, respectively,by graft rejection by the host immune system and by attack on the hostby donor T cells. These diseases are a significant barrier totransplantation systems such as organ transplantations and bone marrowreconstitutions. Other contemplated immune mediated diseases includedelayed-type hypersensitivity (DTH) which is associated with contactantigens such as poison ivy and poison oak and various chemicals, aswell as tuberculosis, leprosy, leishmaniasis, deep fungal infections,etc.

In one embodiment, any autoimmune disease can be treated by the presentmolecular weight markers so long as the contemplated marker binds to anMHC class II protein that has been associated with the autoimmunedisease. One aspect of this embodiment provides a method which includesselecting a molecular weight marker that inhibits binding of anantigenic peptide to an MHC class II protein, for example, a methodwhich further comprises selecting the molecular weight marker thatinhibits class II-specific T cell responses to an MHC class IIprotein-peptide complex, and a method wherein the antigenic peptide isassociated with an autoimmune disease; in another embodiment of theinvention, a method is provided wherein the MHC class II protein isassociated with an autoimmune disease.

In another embodiment, the method for treating an autoimmune disease ina mammal further involves inhibiting the proliferation or function of Tcells which are responsive to an autoantigen. RA is a T cell-mediatedautoimmune disease which can be treated with the present polypeptides.The pathological process of autoimmune diseases and immune rejection ismediated by T cells. Upon binding to and recognition of an antigen, Tcells proliferate, secrete cytokines and recruit additional inflammatoryand cytotoxic cells to the site. The present molecular weight markersprevent T cell proliferation and T cell functions such as cytokinesecretion and recruitment of inflammatory and cytotoxic cells to thesite. When the autoimmune disease is an arthritic condition theautoantigen can be collagen, and the present molecular weight markerscan inhibit the proliferation and function of collagen-responsive Tcells.

In another embodiment, the method for treating an autoimmune disease ina mammal involves binding the molecular weight marker to an antigenpresenting cell such as a macrophage, a dendritic cell of the lymphoidtissue or an epidermal cell. The proliferation and functions of a T cellare activated when an appropriate antigen is presented to it. By bindingto antigen presenting cells, the present molecular weight markers mayblock or otherwise interfere with T cell activation.

In yet another embodiment, the method for treating an autoimmune diseasein a mammal involves binding the molecular weight marker to a majorhistocompatibility complex class II protein which is associated with anautoimmune disease. The Class II MHC proteins are expressedpredominantly on the surfaces of B lymphocytes and antigen presentingcells such as macrophages. These Class II MHC proteins have apeptide-binding cleft which is the site at which antigenic peptides arepresented to T cells. When the present polypeptides bind to a majorhistocompatibility complex class II protein, those polypeptides canblock or otherwise interfere with antigen presentation and/or T cellactivation.

In another embodiment, the method for treating an autoimmune disease ina mammal involves binding the molecular weight marker to Copolymer1-reactive B cell antibodies, and/or Copolymer 1-reactive T cells.Copolymer 1-reactive T_(H)2/3T cells facilitate the therapeutic effectsof Copolymer 1. When binding to Copolymer 1-reactive T cells, thepresent molecular weight markers stimulate those T cells proliferate,secrete antiinflammatory cytokines and enhance the therapeutic benefitsof treatment by the present methods. According to the present invention,the present molecular weight markers also bind to autoantigen-reactiveantibodies which may block the antibody from attacking the targettissue, thereby helping to prevent the autoimmune disease fromprogressing.

The present molecular weight markers may be administered by anyconvenient route. In one embodiment the present molecular weight markerscan be administered by injection to facilitate delivery to the tissuesaffected by the autoimmune disease. Thus, the present molecular weightmarkers may, for example, be injected, ingested, inhaled, or topicallyapplied. The subject molecular weight markers may be incorporated into acream, solution or suspension for topical administration. The presentmolecular weight markers are preferably administered orally, topicallyor by injection without addition of an adjuvant.

Useful Kits of the Invention—In an embodiment of the invention, a kit isprovided for assaying the binding of an analyte to an MHC protein, whichincludes a water-soluble MHC protein, for example which has beenrecombinantly produced in a non-mammalian cell, and a means fordetection of the bound analyte on the MHC protein, and instructions foruse. The MHC protein used in the kit is an MHC class II protein selectedfrom the group consisting of an MHC class II HLA-DR1 protein, an MHCclass II HLA-DR2 protein and an MHC class II HLA-DR4 protein. The kitcan further comprise an autoantigenic peptide. A kit of the inventioncan be used, for example, to test binding of a molecular weight markerof the invention to an MHC class II or inhibition of MHC binding of anautoantigenic peptide.

In a preferred embodiment, the MHC class II protein is produced in aninvertebrate or a microbial cell, such as an insect cell or a yeast celland is therefore devoid of bound peptide in the antigen cleft. The meansfor detecting binding of the analyte to the MHC protein can be anyradioactive, fluorimetric, chemiluminescent, enzymatic or colorimetricmeans known to one of ordinary skill in the art. In a preferredembodiment, the MHC protein is a class II HLA-DR1 or HLA-DR4 protein.Examples of preferred autoantigenic peptide to be included are acollagen II peptide, a peptide derived from myelin basic protein, myelinoligodendrite protein, or a peptide from another protein implicated inan autoimmune disease.

The examples which follow describe the invention in detail with respectto showing how certain specific representative embodiments thereof canbe made, the materials, apparatus and process steps being understood asexamples that are intended to be illustrative only. In particular, theinvention is not intended to be limited to the methods, materials,conditions, process parameters, apparatus and the like specificallyrecited herein.

Throughout this application, various publications, patents, and patentapplications have been referred to. The teachings and disclosures ofthese publications, patents, and patent applications in their entiretiesare hereby incorporated by reference into this application to more fullydescribe the state of the art to which the present invention pertains.

It is to be understood and expected that variations in the principles ofinvention herein disclosed may be made by one skilled in the art and itis intended that such modifications are to be included within the scopeof the present invention.

The following examples further illustrate the invention.

EXAMPLE 1 Physical Properties of TV-Markers

Solid Phase Synthesis

Seven molecular weight markers were made with molecular weights rangingfrom about 3700–12000 daltons in the laboratory of Prof. M. Fridkin(Weizmann Institute of Science) (Table 2). These markers are referred toas TV-markers. The individual peptides were assigned a name TV-##, where## is the number of amino acid residues (e.g. TV-35 is the 35-mermarker). The amino acid composition of these markers meets glatirameracetate specifications (Table 2).

TABLE 2 Ala Glu Tyr Lys TV-35 - Peptide with a molecular weight = 3757daltons Number of residues 15 5 3 12 Molar fraction 0.429 0.143 0.0860.343 TV-45 - Peptide with molecular weight = 4790 daltons Number ofresidues 20 6 4 15 Molar fraction 0.444 0.133 0.089 0.333 TV-56 -Peptide with a molecular weight = 6008 daltons Number of residues 24 8 519 Molar fraction 0.429 0.143 0.089 0.339 TV-66 - Peptide with amolecular weight = 7040 daltons Number of residues 29 9 6 22 Molarfraction 0.439 0.136 0.091 0.333 TV-77 - Peptide with a molecular weight= 8259 daltons Number of residues 33 11 7 26 Molar fraction 0.429 0.1430.091 0.338 TV-86 - Peptide with a molecular weight = 9220 daltonsNumber of residues 37 12 8 29 Molar fraction 0.430 0.140 0.093 0.337TV-109 - Peptide with a molecular weight = 11727 daltons Number ofresidues 46 15 10 38 Molar fraction 0.422 0.138 0.092 0.349

FIGS. 1 a-1, 1 a-2, 1 a-3, 1 b-1, 1 b-2, 1 b-3, 1 c-1, 1 c-2, 1 c-3 1d-1, 1 d-2 and 1 d-3 provide the distribution of alanine, lysine,glutamic acid and tyrosine, respectively, in the TV-markers described inTable 2. The amino acid position is defined by the X-axis, with thefirst amino acid corresponding to the C-terminal position. The presenceof an amino acid is indicated by a vertical bar at the indicated aminoacid position.

Confirmation of Mass and Sequence

Mass Spectroscopy—Polypeptide samples were analyzed immediately aftertheir synthesis using a VG platform mass spectrophotometer equipped withan electronspray ion source. Several months later the analysis wasrepeated at TEVA using a PE-Sciex AP1300 mass spectrophotometer equippedwith an electronspray ion source (Table 3, first preparation). Theseresults indicate that each polypeptide TV-marker has a single, maincomponent with the intended molecular mass.

TABLE 3 Mass Spectroscopy of Sequence-Defined Polypeptides DeterminedDetermined Designed molecular mass - molecular mass - molecular massfirst preparation second preparation Polypeptide (daltons) (daltons)(daltons) TV-35 3757 3757 3757 TV-45 4790 4790 4790 TV-56 6008 6008 6008TV-66 7040 7041 7040 TV-77 8259 8259 8259 TV-86 9220 9220 9220 TV-109*11727 11728 11727 *The 109-mer was further purified by fractionation ona reversed-phase column. Three fractions were collected and fractionnumber 2 was designated for calibration purposes and referred to asTV-109.

A second batch of markers was prepared. Mass spectroscopy confirmed thatthe polypeptides of the second preparation were identical to thepolypeptides of the first preparation (Table 3, second preparation). Thesimilarity between the two preparations was also confirmed bychromatography on Superose 12. Each of the markers eluted with a sharppeak at a distinct retention time, regardless of the batch analyzed.Hence, the TV-markers of the present invention can be synthesized withreproducible mass.

Edman degradation—The intended sequence of the polypeptides wasconfirmed by Edman degradation analysis of the first preparation.

Characterization of the Polypeptides

Circular dichroism—Structural similarity between the molecular weightmarkers and glatiramer acetate is a pre-requisite for an appropriatecalibration of a molecular sizing column. Differences in polypeptidestructure may result in different hydrodynamic size and consequently inaltered retention time in the chromatographic system. The ellipticity,determined by circular dichroism, serves as a measure of the secondarystructure of a polypeptide. When the ellipticity of the molecular weightmarkers and glatiramer acetate is similar, the structures of the twowill be similar.

The molar ellipticity of the polypeptides was determined on a Jobin-YvonCD spectrophotometer. FIG. 2 and Table 4 show that the extent of molarellipticity correlated with the molecular weight of the polypeptide. Theshortest peptide exhibited the lowest ellipticity value. The molarellipticity of the new markers was of the same order of magnitude asthose of the currently used glatiramer acetate molecular weight markers.Note that while the exact molecular weight for the TV-markers wasplotted, the average-by-number molecular weight for the glatirameracetate was used in the plot.

Thus, the new markers and glatiramer acetate possess similar structuresand are therefore suitable for use as molecular weight markers for newpreparations of glatiramer acetate.

TABLE 4 Molecular Ellipticity MW M-ellip. MW marker (daltons) (210 nm)TV-markers TV-35 3757 −1.5367 TV-45 4790 −2.1651 TV-56 6008 −3.9658TV-66 7040 −3.5172 TV-77 8259 −4.8365 TV-86 9220 −5.4546 TV-109 11727−6.818 glatiramer acetate BD 743 3700 −2.0186 BD 714 5600 −4.4182 BD 6816600 −5.2019 BD 677 7000 −6.0153 56895 8000 −6.9062 90995 8500 −9.1736BD 656 8900 −8.8576

These analytical data indicate that the synthesized TV-markerpolypeptides exhibit a substantial degree of similarity to the currentlyused glatiramer acetate molecular weight markers. The amino acid contentis within glatiramer acetate specifications. The new polypeptides andthe glatiramer acetate molecular weight markers have similar secondarystructure, expressed as molar ellipticity. Consequently, TV-markers areexpected to migrate or elute in a gel-permeation-chromatographic (GPC)system, such as Superose 12, like a glatiramer acetate preparation.

EXAMPLE 2 Superose 12 Column Calibration with TV-Markers

TV-markers and a glatiramer acetate preparation are expected todemonstrate a similar correlation between relative retention time (RRT)and log molecular weight. The TV-markers were chromatographed on severalSuperose 12 columns. The peak retention time for each of thepolypeptides was recorded. The linear correlation between Log MolecularWeight (MW) and the Relative Retention Time (RRT) was calculated asfollows: RRT=B1+B2×LogMW (see FIG. 3 a and Table 5).

The recently introduced Millennium-based data acquisition system (WatersCorp., Milford, Mass.) provides integrated calibration of GPC columns.The algorithm for the calibration is based on the retention time and isgiven by the equation:Log MW=A+B×RT or MW=10^((A+B×RT))where MW is the molecular weight, RT is the retention time, A and B,respectively, are the intercept and the slope of the calculatedregression function (FIG. 3 b, Table 5).

The results obtained by this algorithm are practically identical tothose obtained with the currently applied algorithm, based on RRT. Inthe effort to automate procedures, the Millennium-based data acquisitionsystem was employed to perform the calibration using the TV-markers. Theanalytical methods were updated accordingly.

A good correlation (r²>0.98) was obtained between log MW and RRT,although the points do not distribute evenly around the regression line.This distribution is due to the differences in the ellipticity of thevarious markers, as is also observed for the glatiramer acetate. Thesomewhat deviant-from-linearity low molecular weight marker cannot beexcluded because the regression must cover values down to 2500 daltonsfor the first standard deviation (+1 SD) distribution parameter. This isa general trait of all shorter peptides—they are less helical andmore-linear.

For the calibration based on the glatiramer acetate molecular weightmarkers, the intercept (B1) and slope (B2) were, respectively, 1.7415and −0.2784. This compares favorably with the calibration valuesobtained with TV-markers (B1=1.6996; B2=−0.2705). The molecular weightsobtained using the two calibration sets within the specification rangediffered by, typically, not more than 20% in the low molecular weightrange and by not more than 12% in the RRT specification range of thepeak (average molecular weight). This relatively small differencesupports the claim that these markers can replace the currently usedglatiramer acetate molecular weight markers without significant changein the reported molecular weight values.

TABLE 5a Calibration by glatiramer acetate MW-markers Marker MW LOG MWPEAK RT RRT* TV-35 3757 3.575 28.97 0.728 TV-45 4790 3.68 27.96 0.703TV-56 6008 3.779 27.12 0.682 TV-66 7040 3.848 26.32 0.662 TV-77 82593.917 25.56 0.643 TV-86 9220 3.965 24.93 0.627 TV-109 11727 4.069 23.570.593 INTERCEPT **A 6.2516 ***B1 1.6996 SLOPE B −0.0918 B2 −0.2705 r²0.9927 0.9923 *RRT = RT/RTAcetone **calculated according Millenniumequation: log MW = A + B × RT ***calculated according to equation: RRT =B₁ + B₂ × log MW

Calibration based on TV-markers was compared to calibration based onglatiramer acetate molecular weight markers (Table 5b). The twocalibrations were compared by calculating molecular weight values foreach calibration set in the RRT range of 0.5 to 0.8. The TV-markercalibration set included a fraction of TV-109 which was purified byreversed phase chromatography prior to use for column calibration.

TABLE 5b Column Calibration by TV-markers Glatir. Ac. TV (0.1)Difference RT* (MW1) (MWm) (MWm-MW1) RRT (min) Daltons Daltons Daltons %0.5 19.89 28800 26700 −2100 −7.3% 0.51 20.28 26500 24500 −2000 −7.5%0.52 20.68 24400 22600 −1800 −7.4% 0.53 21.08 22500 20700 −1800 −8.0%0.54 21.48 20700 19100 −1600 −7.7% 0.55 21.87 19000 17500 −1500 −7.9%0.56 22.27 17500 16100 −1400 −8.0% 0.57 22.67 16100 14800 −1300 −8.1%0.58 23.07 14900 13600 −1300 −8.7% 0.59 23.46 13700 12500 −1200 −8.8%0.6 23.86 12600 11500 −1100 −8.7% 0.61 24.26 11600 10600 −1000 −8.7%0.62 24.66 10700 9700 −1000 −9.3% 0.63 25.06 9800 9000 −800 −8.2% 0.6425.45 9000 8200 −800 −8.9% 0.65 25.85 8300 7600 −700 −8.4% 0.66 26.257700 7000 −700 −9.1 0.67 26.65 7100 6400 −700 −9.9 0.68 27.04 6600 6900−600 −9.2% 0.69 27.44 6000 5400 −600 −10.0% 0.70 27.84 5500 5000 −500−9.1% 0.71 28.24 5100 4600 −500 −9.8% 0.72 28.63 4700 4200 −500 −10.6%0.73 29.03 4300 3900 −400 −9.3% 0.74 29.43 4000 3600 −400 −10.0% 0.7529.83 3600 3300 −300 −8.3% 0.76 30.23 3400 3000 −400 −11.8% 0.77 30.623100 2800 −300 −9.7% 0.78 31.02 2800 2500 −300 −10.7% 0.79 31.42 26002300 −300 −11.5% 0.80 31.82 2400 2100 −300 −12.5%

Purity of TV markers—Three of the markers (TV-66, TV-77 and TV-86) werefurther purified by reversed phase chromatography. Three fractions wereobtained for each marker. The middle fraction containing the majorportion of the peak was chromatographed on the Superose 12 system incomparison to the unfractionated markers (Table 6). TV markers were sizechromatographed without purification (Regular) and after purification byreversed-phase chromatography (Purified). Peak retention times weredetermined and the differences were calculated. The peak retention timeremained unaffected by the degree of purity. Therefore, the finalproduct of the synthesis is useful for accurate calibration and extrapurification is not required.

TABLE 6 Effect of Purification on Retention Time Retention Time (RT)(min) Difference TV-marker Regular Purified (%) TV-66 26.200 26.233−0.13% TV-77 25.450 25.450 0.00% TV-86 24.867 24.850 0.07%

Consistency in reported values (Cross-validation)—Six batches ofglatiramer acetate, manufactured in 1993 and 1994, were reanalyzed byGPC calibrated with the TV-markers. Their average molecular weight andthe molecular weight distribution was compared to the values reported atthe time of their release. Table 7 shows a comparison of molecularweight data from the original certificate of analysis and molecularweight data obtained using a Superose 12 column calibrated withTV-markers. The differences in reported values are typically less than10%.

TABLE 7 Comparison of Molecular Weight Determinations MW MW % Cop 1preparation Millennium CoA difference 00193 average 10250 9900 −3.5% −1SD 20950 19100 −9.7% +1 SD 51000 4800 −6.3% 00594 average 6700 6550−2.3% −1 SD 15700 15100 −4.0% +1 SD 3600 3400 −5.9% 00993 average 92008600 −7.0% −1 SD 18500 17350 −6.9% +1 SD 4700 4400 −6.8% 04194 average6100 6150 0.8% −1 SD 12600 12500 −0.8% +1 SD 3200 3200 0.0% 01793average 8800 8300 −6.0% −1 SD 18100 17300 −4.6% +1 SD 5200 4750 −9.5%05494 average 8100 8300 2.4% −1 SD 17800 17450 −2.0% +1 SD 4100 41000.0%

Stability of markers in solution—TV-markers were chromatographed fourtimes over a period of 24 hours. All markers were kept as solutions atroom temperature and were analyzed at 8 hour intervals. Table 8 showsthe peak retention time measured for the TV-markers at each of the fourtime points. At a concentration of 0.1 mg/ml, the TV-markers were stablein solution for at least 24 hours at room temperature.

TABLE 8 Stability of TV-markers in solution at room temperature. PeakRetention Time (min) Average RSD TV-35 29.883 29.883 29.900 29.95029.904 0.106% TV-45 28.933 28.917 28.917 28.933 28.925 0.032% TV-5628.250 28.217 28.283 28.250 28.250 0.095% TV-66 27.400 27.350 27.43327.433 27.404 0.143% TV-77 26.750 26.700 26.750 26.783 26.746 0.128%TV-86 26.117 26.100 26.150 26.150 26.129 0.095% TV-109Fr 11 24.78324.850 24.883 24.850 24.842 0.169%

In addition, solutions of the markers were stored for up to 3½ monthsunder various storage conditions (2–8° C., −10 to −20° C., with/withoutazide). TV-markers are stable for at least 3 months when stored asfrozen solutions (Table 9). As a precaution it was decided to allowstorage of frozen solutions for two months.

Lyophilized TV-markers are stable for at least two years according toaccumulated stability data.

TABLE 9 Stability of TV-markers at −10° to −20° C. Date of calibration:22-May-97 09-Jul-97 04-Sep-97 Interval (days) — 48 105 Marker MW RT RTRT TV-35 3757 28.867 28.867 28.967 TV-45 4790 27.833 27.917 27.950 TV-566008 27.076 27.133 27.100 TV-66 7040 26.233 26.317 26.300 TV-77 825925.467 25.617 25.550 TV-86 9220 24.883 25.017 24.950 TV-109 11727 23.50023.650 23.583

Summary of calibration data—Overall, the TV-markers were analyzed 53times in two laboratories. A summary of the data is presented in FIG. 4and Table 10. The differences observed among the individual runs (FIG.4) reflect variations between columns rather than differences betweenthe participating laboratories. This is indicated in FIG. 4 by the useof different symbols for some of the runs. Calibration constants inTable 10 were calcualted using the Millennium equation for data obtainedfor 53 calibration sets injected into 16 columns.

TABLE 10 Calibration constants obtained in Plantex and Abic Labs RT RTMarker MW Mean SD RSD % Min Max Mean − SD Mean + SD TV-35 3757 29.690.463 1.6% 28.85 30.35 28.30 31.08 TV-45 4790 28.72 0.481 1.7% 27.8829.40 27.28 30.16 TV-56 6008 27.99 0.520 1.9% 27.08 28.77 26.43 29.55TV-66 7040 27.19 0.526 1.9% 26.26 27.96 25.61 28.77 TV-77 8259 26.490.550 2.1% 25.51 27.33 24.84 28.14 TV-86 9220 25.89 0.556 2.1% 24.8926.72 24.22 27.56 TV-109 11727 24.56 0.557 2.3% 23.53 25.41 22.89 26.23Intercept (A) 6.4706 0.1220 1.9% 6.2561 6.6500 6.1046 6.8366 Slope (B)−0.0969 0.0032 −3.3% −0.1014 −0.0919 −0.1064 −0.0873 r² 0.9901 0.00220.2% 0.9868 0.9828 0.9835 0.9967

Molecular weight distribution of a glatiramer acetatepreparation—Molecular weight was determined for a batch of glatirameracetate (BN 90995). Table 11 summarizes data obtained from 16determinations on TV-marker-calibrated columns.

TABLE 11a RT of glatiramer acetate (BN 90995) Average SD RSD % Peak26.208 0.434 1.66 −2SD (2.5%) 19.865 0.528 2.66 −1SD (16%) 22.578 0.4772.11 +1SD (84%) 28.934 0.324 1.12

TABLE 11b RRT of glatiramer acetate (BN 90995) Average SD RSD % Peak0.664 0.014 2.09 −2SD (2.5%) 0.503 0.016 3.09 −1SD (16%) 0.572 0.0152.54 +1SD (84%) 0.733 0.011 1.53

TABLE 11C MW (Daltons) of glatiramer acetate (BN 90995) Date Average SDRSD % Peak 7459 146 1.95 −1SD (16%) 16622 466 2.80 +1SD (84%) 4089 771.89

The application of a molecular weight and sequence-defined set ofmarkers for the calibration of the Superose 12 column has severaladvantages over the currently used glatiramer acetate molecular weightmarkers.

First, the use of solid phase synthesis assures consistency among thevarious preparations of each batch. Mass spectroscopy results (Table 3)confirmed the reproducibility of the synthesis. This consistencyprovides improved accuracy in molecular weight determinations.

Second, the current calibration is based on the determination of the RRTat 50% of the peak area for each of the glatiramer acetate molecularweight markers. The new markers elute as sharp peaks. Their use incalibration is more accurate than the calculated retention time at 50%of the area of a broad peak.

Third, the use of markers having molecular weights defined bypredetermined sequence precludes any uncertainty which might accompanythe use of markers whose molecular weight is determined by inexactmeasurement of physical properties.

Fourth, the calibration procedure facilitates normalization of columnsfor molecular weight determinations, regardless of minor changes betweencolumn lots, age or instrumentation.

EXAMPLE 3 Biological Activity of TV-Markers

Reactivity of TV-markers with monoclonal antibodies to Cop 1.—Table 12shows the binding of anti-Cop 1 monoclonal antibodies to TV-markers.TV-markers and reference Glat production batches were tested. Microtiterwells were coated with 2 μg/ml antigen. Values are counts per minute(cpm) of ¹²⁵I-goat anti-mouse IgG bound to the monoclonal antibodies.Antibody binding to each TV-marker is compared to antibody binding toCop 1 reference standard.

TABLE 12 Reactivity of TV-markers and Cop 1 with mAbs in RIA Binding ofmAb cpm (% Cop 1 binding) anti-Cop-1 anti-Cop-1 anti-Cop-1 CoatingAntigen (3-3-9) (3-1-45) (5-7-2) PBS  1384  315  521 03494 14860 2058710513 (glatiramer acetate) 55296 13705 (91) 17189 (83) 8683 (82)(glatiramer acetate) 55396 13458 (90) 17564 (85) 9142 (86) (glatirameracetate) TV-35  1176 (0)  343 (0)  657 (1) (TV-marker) TV-56  1614 (2) 1581 (6) 9584 (91) (TV-marker) TV-77  2265 (6)  2152 (9) 4259 (37)(TV-marker) TV-86  1625 (2)  1606 (6) 8140 (76) (TV-marker)

Reactivity with Cop 1 specific T cells.—T cells lines which can bestimulated with GLAT copolymer were used to test stimulatory activity ofTV-markers in comparison to regular GLAT copolymer production batches(Table 13). As above, the activities of TV-markers were tested for invitro. The proliferation of various mouse and human T cell lines wasdetermined in response to peptides in culture. The cell lines included:BALB/c-Ts-Cop-1, a tempreature-sensitive line derived from BALB/c mice;L-22-1, a tempreature-sensitive clone derived from F₁ mice; SC-103 andSC-14: human Cop 1 specific T cell clones. Proliferation was determinedby measuring ³H-thymidine uptake by the T cell lines cultured with 10 μgof GLAT copolymer or TV-marker.

Glatiramer acetate batches were stimulatory. TV-markers were also foundto stimulate two of the four T cell lines, although not as strongly.TV-markers are recognized by both mouse and human T cells specific toglatiramer acetate. This confirms that there is amino acid sequencesimilarity and T cell epitope similarity among glatiramer acetate andTV-markers.

TABLE 13 Reactivity of glatiramer acetate and TV-markers with glatirameracetate specific T cell lines ³H-Thymidine incorporation cpm (% Cop 1)BALB/c- Antigen Ts-Cop-1 L-22-1 SC-103 SC-14 PBS  588  207  342  76003494 32643 16395 8709 3091 (glatiramer acetate) 55296 35820 (110) 17315(106) 7148 (81) 2973 (95) (glatiramer acetate) 55396 34281 (105) 17211(105) 7019 (80) 3253 (107) (glatiramer acetate) TV-35  9465 (28)  225(0)  438 (0)  884 (0) (TV-marker) TV-56 19545 (59)  232 (0)  237 (0)3495 (117) (TV-marker) TV-77 17367 (52)  300 (1)  327 (0) 2701 (83)(TV-marker) TV-86 14694 (44)  418 (1)  298 (0) 2284 (65) (TV-marker)

Blocking of Experimental Allergic Encephalomyelitis—To test thephysiological activity of TV-markers, protection from experimentalallergic encephalomyelitis (EAE) was investigated in mice. Injection ofCopolymer 1 in complete Freund's adjuvant together with theencephalitogen can block EAE essentially as described in Aharoni et al.,17 EUR. J. IMMUNOL. 23 (1993). Other researchers have observed that thetherapeutic effect of Copolymer 1 in multiple sclerosis patients is alsoassociated with the induction of T_(H)2 cells. Lahat et al., 244 J.NEUROL. 129 (1997). In this example, EAE is blocked by differentpolypeptides of the present invention.

Induction of EAE—Two to three month old female (SJL/JxBALB/c)FI mice areinjected in all four footpads with mouse spinal cord homogenate (3.5mg/mouse) emulsified in a 1:1 ratio in complete Freund's adjuvant (CFA)supplemented with 4 mg/ml mycobacterium tuberculosis H37Ra. Pertussistoxin (0.25 ml, 250 ng, Sigma) is injected intravenously, immediatelyafter and 48 hr later. Mice are examined daily from day 10 postinduction for clinical signs of EAE which were scored on a 0–5 scale asdescribed in Lando et al., 123 J. IMMUNOL. 2156 (1979).

EAE blocking by injection with complete adjuvant—Each antigen beingtested was included in the encephalitogenic inoculum. Table 14 shows theincidence of EAE in animals which received the encephalitogenic inoculumsupplemented with a TV-marker or glatiramer acetate and in animals whichreceived only the encephalitogenic inoculum. Also shown is the meanonset of EAE in animals which were not protected. Disease intensity isscored daily in mice with a score of zero (0=healthy) to five (5=dead).The onset is determined as the day an animal exhibits a disease score ofat least one (1).

TABLE 14 Protection from EAE by TV-markers Blocking Mean Onset AntigenIncidence Mean Score (days) % Blocking None (control) 10/10  4.9 11.3 —TV-45 0/10 0 — 100 TV-66 6/10 2.8 11.7 40 TV-77 1/9  0.2 14.0 89 TV-863/10 0.7 12.0 70 TV-109 0/10 0 — 100 03494 0/10 0 — 100 55396 0/10 0 —100

1. In a process for obtaining a pharmaceutical product containing amixture of polypeptides, each of which consists essentially of alanine,glutamic acid, tyrosine and lysine, wherein the mixture has an averagemolecular weight from 4000 to 13,000 Daltons and in the mixture themolar fraction of alanine is 0.427, of glutamic acid is 0.141, of lysineis 0.337 and of tyrosine is 0.093 and wherein the process includesdetermining the molecular weight distribution of a batch of an aqueousmixture of polypeptides, each of which consists essentially of alanine,glutamic acid, tyrosine and lysine, using a gel permeationchromatography column to determine whether the mixture has an averagemolecular weight from 4000 to 13,000 Daltons for inclusion in thepharmaceutical product, the improvement comprising calibrating themolecular weight obtained using the gel permeation chromatography columnby subjecting a plurality of molecular weight markers, each of which isa polypeptide consisting essentially of alanine, glutamic acid, tyrosineand lysine and having a predetermined amino acid sequence, tochromatography on the column to establish a relationship betweenretention time on the column and molecular weight.
 2. The process ofclaim 1, wherein the gel permeation chromatography column comprises across-linked agarose-based medium, with an exclusion limit of 2×10⁶Daltons, an optimal separation range of 1000 to 3×10⁶ Daltons, and abead diameter of 20–40 μm.
 3. The process of claim 1, wherein in themolecular weight markers, the molar fraction of alanine is 0.38 to 0.5,of glutamic acid is 0.13 to 0.15, of tyrosine is 0.08 to 0.10 and oflysine is 0.3 to 0.4.
 4. The process of claim 3, wherein in themolecular weight markers, the molar fraction of alanine is 0.422 to0.444, of glutamic acid is 0.133 to 0.143, of tyrosine is 0.086 to 0.093and of lysine is 0.333 to 0.349.
 5. The process of claim 1, wherein oneof the molecular weight markers is selected from the group consisting of(SEQ ID NO: 1) AKKYAKKEKAAKKAYKKEAKAKAAEAAAKEAAYEA; (SEQ ID NO: 2)AKKYAKKAKAEKAKKAYKAAEAKKAAKYEKAAAEKAAAKEAAYEA; (SEQ ID NO: 3)AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAEAKYKAEAAKAAAKE AAYEA; (SEQ ID NO:4) AKKYAKKEKAYAKAKKAEAKAAKKAKAEAKKYAKAAKAEKKEYAAAEAKYK; AEAAKAAAKEAAYEA;(SEQ ID NO: 5) AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAKAEAKKYAKAAKAEKKEYAAAEAKYKAEAAKAAAKEAAYEA; (SEQ ID NO: 6)AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAKAEAKKYAKAAKAEKKEYAAAEAKYKAEAAKKAYKAEAAKAAAKEAAYEA; and (SEQ ID NO: 7)AKKYAKKAEKAYAKKAKAAKEKKAYAKKEAKAYKAAEAKKKAKAEAKKYAKEAAKAKKEAYKAEAKKYAKAAKAEKKEYAAAEAKKAEAAKAYKAEAAKAAA KEAAYEA,

wherein A represents alanine, K represents lysine, Y representstyrosine, and E represents glutainic acid.
 6. The process of claim 1,wherein the plurality of molecular weight markers is (SEQ ID NO: 1)AKKYAKKEKAAKKAYKKEAKAKAAEAAAKEAAYEA; (SEQ ID NO: 2)AKKYAKKAKAEKAKKAYKAAEAKKAAKYEKAAAEKAAAKEAAYEA; (SEQ ID NO: 3)AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAEAKYKAEAAKAAAKE AAYEA; (SEQ ID NO:4) AKKYAKKEKAYAKAKKAEAKAAKKAKAEAKKYAKAAKAEKKEYAAAEAKYK; AEAAKAAAKEAAYEA;(SEQ ID NO: 5) AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAKAEAKKYAKAAKAEKKEYAAAEAKYKAEAAKAAAKEAAYEA; (SEQ ID NO: 6)AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAKAEAKKYAKAAKAEKKEYAAAEAKYKAEAAKKAYKAEAAKAAAKEAAYEA; and (SEQ ID NO: 7)AKKYAKKAEKAYAKKAKAAKEKKAYAKKEAKAYKAAEAKKKAKAEAKKYAKEAAKAKKEAYKAEAKKYAKAAKAEKKEYAAAEAKKAEAAKAYKAEAAKAAA KEAAYEA,

wherein A represents alanine, K represents lysine, Y representstyrosine, and E represents glutainic acid.
 7. The process of claim 1,wherein the pharmaceutical product is lyophilized.
 8. A process forobtaining a pharmaceutical product containing a mixture of polypeptides,each of which consists essentially of alanine, glutamic acid, tyrosineand lysine, wherein the mixture has an average molecular weight from4000 to 13,000 Daltons and in the mixture the molar fraction of alanineis 0.427, of glutainic acid is 0.141, of lysine is 0.337 and of tyrosineis 0.093, which comprises obtaining a batch of a mixture ofpolypeptides, each of which consists essentially of alanine, glutamicacid, tyrosine and lysine; determining the average molecular weight ofthe mixture of polypeptides in the batch using a molecularweight-calibrated gel permeation chromatography column; and including inthe pharmaceutical product the mixture if the mixture is determined tohave an average molecular weight from 4000 to 13,000 Daltons, whereinthe gel permeation chromatography column is calibrated by subjecting aplurality of molecular weight markers to chromatography on the column toestablish a relationship between the retention time on the column andmolecular weight, wherein each of the markers is a polypeptideconsisting essentially of alanine, glutamic acid, tyrosine and lysineand has a predetermined amino sequence.
 9. The process of claim 8,wherein the gel permeation chromatography column comprises across-linked agarose-based medium, with an exclusion limit of 2×10⁶Daltons, an optimal separation range of 1000 to 3×10⁵ Daltons, and abead diameter of 20–40 μm.
 10. The process of claim 8, wherein in themolecular weight markers, the molar fraction of alanine is 0.38 to 0.5,of glutamic acid is 0.13 to 0.15, of tyrosine is 0.08 to 0.10 and oflysine is 0.3 to 0.4.
 11. The process of claim 10, wherein in themolecular weight markers, the molar fraction of alanine is 0.422 to0.444, of glutamic acid is 0.133 to 0.143, of tyrosine is 0.086 to 0.093and of lysine is 0.333 to 0.349.
 12. The process of claim 8, wherein oneof the molecular weight markers is selected from the group consisting of(SEQ ID NO: 1) AKKYAKKEKAAKKAYKKEAKAKAAEAAAKEAAYEA; (SEQ ID NO: 2)AKKYAKKAKAEKAKKAYKAAEAKKAAKYEKAAAEKAAAKEAAYEA; (SEQ ID NO: 3)AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAEAKYKAEAAKAAAKE AAYEA; (SEQ ID NO:4) AKKYAKKEKAYAKAKKAEAKAAKKAKAEAKKYAKAAKAEKKEYAAAEAKYK; AEAAKAAAKEAAYEA;(SEQ ID NO: 5) AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAKAEAKKYAKAAKAEKKEYAAAEAKYKAEAAKAAAKEAAYEA; (SEQ ID NO: 6)AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAKAEAKKYAKAAKAEKKEYAAAEAKYKAEAAKKAYKAEAAKAAAKEAAYEA; and (SEQ ID NO: 7)AKKYAKKAEKAYAKKAKAAKEKKAYAKKEAKAYKAAEAKKKAKAEAKKYAKEAAKAKKEAYKAEAKKYAKAAKAEKKEYAAAEAKKAEAAKAYKAEAAKAAA KEAAYEA,

wherein A represents alanine, K represents lysine, Y representstyrosine, and E represents glutamic acid.
 13. The process of claim 8,wherein the plurality of molecular weight markers is (SEQ ID NO: 1)AKKYAKKEKAAKKAYKKEAKAKAAEAAAKEAAYEA; (SEQ ID NO: 2)AKKYAKKAKAEKAKKAYKAAEAKKAAKYEKAAAEKAAAKEAAYEA; (SEQ ID NO: 3)AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAEAKYKAEAAKAAAKE AAYEA; (SEQ ID NO:4) AKKYAKKEKAYAKAKKAEAKAAKKAKAEAKKYAKAAKAEKKEYAAAEAKYK; AEAAKAAAKEAAYEA;(SEQ ID NO: 5) AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAKAEAKKYAKAAKAEKKEYAAAEAKYKAEAAKAAAKEAAYEA; (SEQ ID NO: 6)AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAKAEAKKYAKAAKAEKKEYAAAEAKYKAEAAKKAYKAEAAKAAAKEAAYEA; and (SEQ ID NO: 7)AKKYAKKAEKAYAKKAKAAKEKKAYAKKEAKAYKAAEAKKKAKAEAKKYAKEAAKAKKEAYKAEAKKYAKAAKAEKKEYAAAEAKKAEAAKAYKAEAAKAAA KEAAYEA,

wherein A represents alanine, K represents lysine, Y representstyrosine, and E represents glutainic acid.
 14. The process of claim 8,further comprising a step of lyophilizing the mixture of polypeptides.15. A process for determining the average molecular weight of an aqueousmixture of polypeptides, each of which consists essentially of alanine,glutamic acid, tyrosine and lysine, wherein in the mixture the molarfraction of alanine is 0.427, of glutamic acid is 0.141, of lysine is0.337 and of tyrosine is 0.093, which comprises subjecting the mixtureto chromatography on a molecular weight-calibrated gel permeationchromatography column so as to determine the average molecular weight ofthe mixture, wherein the gel permeation chromatography column iscalibrated by subjecting a plurality of molecular weight markers tochromatography on the column to establish a relationship betweenretention time on the column and molecular weight, wherein each of themarkers is a polypeptide consisting essentially of alanine, glutamicacid, tyrosine and lysine and has a predetermined amino acid sequence.16. The process of claim 15, wherein the gel permeation chromatographycolumn comprises a cross-linked agarose-based medium, with an exclusionlimit of 2×10⁶ Daltons, an optimal separation range of 1000 to 3×10⁵Daltons, and a bead diameter of 20–40 μm.
 17. The process of claim 15,wherein in the molecular weight markers, the molar fraction of alanineis 0.38 to 0.5, of glutamic acid is 0.13 to 0.15, of tyrosine is 0.08 to0.10 and of lysine is 0.3 to 0.4.
 18. The process of claim 17, whereinin the molecular weight markers, the molar fraction of alanine is 0.422to 0.444, of glutamic acid is 0.133 to 0.143, of tyrosine is 0.086 to0.093 and of lysine is 0.333 to 0.349.
 19. The process of claim 15,wherein one of the molecular weight markers is selected from the groupconsisting of (SEQ ID NO: 1) AKKYAKKEKAAKKAYKKEAKAKAAEAAAKEAAYEA; (SEQID NO: 2) AKKYAKKAKAEKAKKAYKAAEAKKAAKYEKAAAEKAAAKEAAYEA; (SEQ ID NO: 3)AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAEAKYKAEAAKAAAKE AAYEA; (SEQ ID NO:4) AKKYAKKEKAYAKAKKAEAKAAKKAKAEAKKYAKAAKAEKKEYAAAEAKYK; AEAAKAAAKEAAYEA;(SEQ ID NO: 5) AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAKAEAKKYAKAAKAEKKEYAAAEAKYKAEAAKAAAKEAAYEA; (SEQ ID NO: 6)AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAKAEAKKYAKAAKAEKKEYAAAEAKYKAEAAKKAYKAEAAKAAAKEAAYEA; and (SEQ ID NO: 7)AKKYAKKAEKAYAKKAKAAKEKKAYAKKEAKAYKAAEAKKKAKAEAKKYAKEAAKAKKEAYKAEAKKYAKAAKAEKKEYAAAEAKKAEAAKAYKAEAAKAAA KEAAYEA,

wherein A represents alanine, K represents lysine, Y representstyrosine, and E represents glutaxnic acid.
 20. The process of claim 15,wherein the plurality of molecular weight markers is (SEQ ID NO: 1)AKKYAKKEKAAKKAYKKEAKAKAAEAAAKEAAYEA; (SEQ ID NO: 2)AKKYAKKAKAEKAKKAYKAAEAKKAAKYEKAAAEKAAAKEAAYEA; (SEQ ID NO: 3)AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAEAKYKAEAAKAAAKE AAYEA; (SEQ ID NO:4) AKKYAKKEKAYAKAKKAEAKAAKKAKAEAKKYAKAAKAEKKEYAAAEAKYK; AEAAKAAAKEAAYEA;(SEQ ID NO: 5) AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAKAEAKKYAKAAKAEKKEYAAAEAKYKAEAAKAAAKEAAYEA; (SEQ ID NO: 6)AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAKAEAKKYAKAAKAEKKEYAAAEAKYKAEAAKKAYKAEAAKAAAKEAAYEA; and (SEQ ID NO: 7)AKKYAKKAEKAYAKKAKAAKEKKAYAKKEAKAYKAAEAKKKAKAEAKKYAKEAAKAKKEAYKAEAKKYAKAAKAEKKEYAAAEAKKAEAAKAYKAEAAKAAA KEAAYEA,

wherein A represents alanine, K represents lysine, Y representstyrosine, and E represents glutaxnic acid.
 21. A process for determiningwhether an aqueous mixture of polypeptides, each of which consistsessentially of alanine, glutamic acid, tyrosine and lysine, has anaverage molecular weight from 4000 to 13,000 Daltons, wherein in themixture the molar fraction of alanine is 0.427, of glutamic acid is0.141, of lysine is 0.337 and of tyrosine is 0.093, which processcomprises subjecting the mixture to a calibrated gel permeationchromatography column to determine the average molecular weight of themixture wherein the gel permeation chromatography column is calibratedby subjecting a plurality of molecular weight markers to chromatographyon the column to establish a relationship between retention time on thecolumn and molecular weight, wherein each of the markers is apolypeptide consisting essentially of alanine, glutamic acid, tyrosineand lysine and has a predetermined amino acid sequence.
 22. The processof claim 21, wherein the gel permeation chromatography column comprisesa cross-linked agarose-based medium, with an exclusion limit of 2×10⁶Daltons, an optimal separation range of 1000 to 3×10⁵ Daltons, and abead diameter of 20–40 μm.
 23. The process of claim 21, wherein in themolecular weight markers, the molar fraction of alanine is 0.38 to 0.5,of glutamic acid is 0.13 to 0.15, of tyrosine is 0.08 to 0.10 and oflysine is 0.3 to 0.4.
 24. The process of claim 23, wherein in themolecular weight markers, the molar fraction of alanine is 0.422 to0.444, of glutamic acid is 0.133 to 0.143, of tyrosine is 0.086 to 0.093and of lysine is 0.333 to 0.349.
 25. The process of claim 21, whereinone of the molecular weight markers is selected from the groupconsisting of (SEQ ID NO: 1) AKKYAKKEKAAKKAYKKEAKAKAAEAAAKEAAYEA; (SEQID NO: 2) AKKYAKKAKAEKAKKAYKAAEAKKAAKYEKAAAEKAAAKEAAYEA; (SEQ ID NO: 3)AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAEAKYKAEAAKAAAKE AAYEA; (SEQ ID NO:4) AKKYAKKEKAYAKAKKAEAKAAKKAKAEAKKYAKAAKAEKKEYAAAEAKYK; AEAAKAAAKEAAYEA;(SEQ ID NO: 5) AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAKAEAKKYAKAAKAEKKEYAAAEAKYKAEAAKAAAKEAAYEA; (SEQ ID NO: 6)AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAKAEAKKYAKAAKAEKKEYAAAEAKYKAEAAKKAYKAEAAKAAAKEAAYEA; and (SEQ ID NO: 7)AKKYAKKAEKAYAKKAKAAKEKKAYAKKEAKAYKAAEAKKKAKAEAKKYAKEAAKAKKEAYKAEAKKYAKAAKAEKKEYAAAEAKKAEAAKAYKAEAAKAAA KEAAYEA,

wherein A represents alanine, K represents lysine, Y representstyrosine, and E represents glutarnic acid.
 26. The process of claim 21,wherein the plurality of molecular weight markers is (SEQ ID NO: 1)AKKYAKKEKAAKKAYKKEAKAKAAEAAAKEAAYEA; (SEQ ID NO: 2)AKKYAKKAKAEKAKKAYKAAEAKKAAKYEKAAAEKAAAKEAAYEA; (SEQ ID NO: 3)AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAEAKYKAEAAKAAAKE AAYEA; (SEQ ID NO:4) AKKYAKKEKAYAKAKKAEAKAAKKAKAEAKKYAKAAKAEKKEYAAAEAKYK; AEAAKAAAKEAAYEA;(SEQ ID NO: 5) AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAKAEAKKYAKAAKAEKKEYAAAEAKYKAEAAKAAAKEAAYEA; (SEQ ID NO: 6)AKKYAKKEKAYAKKAEKAAKKAEAKAYKAAEAKKKAKAEAKKYAKAAKAEKKEYAAAEAKYKAEAAKKAYKAEAAKAAAKEAAYEA; and (SEQ ID NO: 7)AKKYAKKAEKAYAKKAKAAKEKKAYAKKEAKAYKAAEAKKKAKAEAKKYAKEAAKAKKEAYKAEAKKYAKAAKAEKKEYAAAEAKKAEAAKAYKAEAAKAAA KEAAYEA,

wherein A represents alanine, K represents lysine, Y representstyrosine, and E represents glutamic acid.